Team:DUT China B/Experiments
1 Construction of expression vectors
The plasmid of pOpt-clover-Paro (donated from professor Kong, DUT, China) was used as backbone for vector construction in our project. The green microalgae Chlamydomonas reinhardtii CC4533 from Chlamydomonas Resource Center (https://www.chlamycollection.org/) was used in our project. We coloned the gene of interest to this vector. The schematic representatives of vectors are shown as the following. In, first intron of the Chlamydomonas RbcS2 gene; RbcS2 T, Chlamydomonas RbcS2 terminator; Paro, Paromycin; HygB, Hygromycin B; VchR，Channelrhodopsins of Volvox；mCerulean3，a kind of cyan fluorescent proteins；Renilla luciferase，luciferin 2-monooxygenase from Renilla reniformis，N-hrluc-Pif3, fusion N-rluc with N-terminal phytochrome interacting factor 3; SP-PhyB-C-hrluc，fusion C-rluc with Phytochrome B.
We also constructed the following constructs for expression in E.coli.N-nanoluc-Spytag-1, the combination of a new N terminal of Guassia luciferase and Spytag; SpyCatcher-C-nanoluc-1，the combination of new C terminal of Guassia luciferase and SpyCatcher；N-nanoluc-Spytag-2, see BBa_K1159201; SpyCatcher-C-nanoluc-2, BBa_K1159200.
2 Transformation and expression 
We introduced the genes of interest into C. reinhardtii CC4533 by electroporation, and obtained stable transformants through the screening of palonmycin or hygromycin B-resistant colonies. The target gene was introduced into E. coli DH5α (TaKaRa, Japan) by heat shock. For details, see Protocol 1 .
3 Measurement of the movement features of C. reinhardtii
A standard C. reinhardtii kinetics measurement protocol was established by measuring the velocity of wild-type C. reinhardtii under blue light and determining the illuminance-velocity function.
4 Characterization of the mutant channel rhodopsin VchR from the Volvox
We characterized the effect of expression through measuring movement features of VchR-expressing C. reinhardtii. Measuring protocols see Protocol 5.
5 Effect of endogenous blue light on the trending movement of C. reinhardtii
5.1 mCerulean3
Observe the fluorescence of mCerulean3 expressing C. reinhardtii and explore the motion characteristics of mCerulean3 transformants.
5.2 Renilla luciferase
For the configuration, preservation and use of coelenterin solution, see protocol 4.
Determination of optimal incubation time of Rluc.
Mix 2900 μL crude enzyme solution and 100 μL coelenterin working solution and then measure the luminescence intensity at 480 nm every 5 min from 0up to 30 min.
Determination of optimal substrate concentration of Rluc.
Mix appropriate amount of crude enzyme solution and the prepared coelenterin working solution, to make sure the working concentration coelenterin are 0, 5, 10, 15, 20 μM, respectively. After incubation for 10 min, measure the luminescence intensity at 480 nm.
Expression of Renilla luciferase in C. reinhardtii
Transfer the pOpt-Rluc-paro plasmid to C. reinhardtii CC4533. 2700 μL of the culture solution was used, and after incubation with 300 μL of coelenterazine for 10 min, the intensity of the emitted light at 480 nm was measured.
5.3 Guassia luciferase
st-1: the combination of new N terminal of Guassia luciferase and Spytag
st-2: the combination of N terminal of Guassia luciferase and Spytag
sc-1: the combination of new C terminal of Guassia luciferase and SpyCatcher
sc-2: the combination of C terminal of Guassia luciferase and SpyCatcher
Directly homogenize st-1, st-2, sc-1, sc-2 engineered bacteria (IPTG induced) and collect supernatant crude enzyme solution by centrifugation. After determining the protein concentration, it is used for luminescence detection. Add 1350 μL each of these two interacting crude enzyme solutions in 3 mL system. Incubate for 10min and wait for the combination of Spytag and SpyCatcher, in order to restore the activity of Nanoluc. Add 300 μL coelenterazine solution and incubate it for 10 min to detect the emission light at 480 nm, so as to determine the activity of these two splitting sites.
5.4 PhyB/Pif3
For the configuration, preservation and use of PCB solution, see protocol 3.
Homogenize SP-PhyB-C-Rluc and N-Rluc-Pif3 engineered bacteria (IPTG induced) and collect supernatant crude enzyme solution by centrifugation. After determining the protein concentration, it is used for luminescence detection. After adding 1200 μL of these two proteins, they were incubated with 100 μL of PCB for 30 min, irradiated with red light for 5 min, and incubated with 300 μL of coelenterazine for 10 min, and the emission wavelength at 480 nm was measured.
Transfer the pOpt_SP-PhyB-C-Rluc_paro, pOpt_N-Rluc-Pif3_Hyg plasmids to Engineering C. reinhardtii CC4533 by electroporation. 2700 μL of the culture solution was taken, and after incubation with 300 μL of coelenterazine for 10 min, the intensity of the emitted light at 480 nm was measured.
We characterized the function of our design through measuring movement features of PhyB/Pif3-engineered C. reinhardtii. Measuring protocols see Protocol.